Study of Methylomes in Aneurinibacillus Species using Third Generation Sequencing Platform

abstract

English

Detection of methylation in bacterial genomes has been crucial in understanding how gene regulation, pathogenesis and adaptation work in bacteria [1]. It helps us understand how genetic and metabolic capabilities can help us understand the industrial potential of the bacteria. Thermophilic members of the Aneurinibacillus genus are gram-positive spore-forming bacteria species recently identified as producers of polyhydroxyalkanoates (PHAs) [2], a precursor for bioplastics. In this study, we utilized Oxford Nanopore Technologies (ONT) sequencing to investigate methylomes in Aneurinibacillus sp. AFN2 across various genomic features. Sequencing was done using the PromethION system with R10.4 flow cells. Genomic annotation was conducted using the Prokka tool [3], for comprehensive gene prediction and functional annotation. The sequences were basecalled using the modified basecalling feature of Dorado, later Modkit was employed to handle modified bases from ONT, specifically for converting modBAM to bedMethyl files using best practices. Out of the Total modifications, 50,808 were found to be in the coding region (CDS) out of which 22,003 were 5mC and 28,805 were 6mA modifications which is around 92% of the total of the total modifications. In the rRNA region, 1,526 5mC sites and 1,534 6mA modification sites were detected while in repeated regions 48 5mC sites and 54 6mA sites, highlighting their potential role in genome stability and regulation. tRNA showed 5 sites of 5mC and 22 sites of 6mA modification as shown in Figure 1. The total of 1065 modifications were not found in any annotated feature. Furthermore, we also calculated the modification density for both methylation types. The density of 5mC modification was found to be around 0.006 per base while the density of 6mA modification was approximately 0.008 per base thus indicating the higher occurrence of 6mA modification in the bacterial genome. These findings determine the widespread methylation pattern and are distributed among various genomic features. Most of the methylation in CDS shows the role played by methylation in gene regulation and expression in the bacteria. The differential distribution of 5mC and 6mA across genomic features can provide insights into various functional roles and regulatory mechanisms within the genome. This study underscores the robustness of ONT sequencing in detecting and quantifying DNA methylation, offering valuable insights into the epigenetic landscape of Aneurinibacillus species

methylation, Aneurinibacillus, Oxford Nanopore, bacterial genomics.

UMAIR, M.; ŘEHÁKOVÁ, V.; BEZDÍČEK, M.; BUCHTÍKOVÁ, I.; OBRUČA, S.; SEDLÁŘ, K.

12. 9. 2024

55

55

1

https://www.fch.vut.cz/chl/konference/program/bookofabstracts-pdf-p269547